Aflatoxin B1 (AFB1) is a prevalent environmental and forage contaminant that poses significant health risks to both humans and livestock due to its toxic effects on various organs and systems. Among its toxicological effects, nephrotoxicity is a hallmark of AFB1 exposure. However, the precise mechanisms underlying AFB1-induced kidney damage in donkeys remain poorly understood. To investigate this, we established a donkey model exposed to AFB1 by administering a diet supplemented with 1 mg AFB1/kg for 30 days. Kidney apoptosis was assessed using TUNEL staining, while gene expression and protein levels of Endonuclease G (EndoG), as well as genes related to endoplasmic reticulum (ER) stress and apoptosis, were quantified by RT-qPCR and Western blotting. Our findings indicate that AFB1 exposure resulted in significant kidney injury, apoptosis, and oxidative stress. Notably, AFB1 exposure upregulated the expression of EndoG and promoted its translocation to the ER, which subsequently induced ER stress and activated the mitochondrial apoptotic pathway. These results suggest that AFB1-induced kidney damage in donkeys is mediated through the oxidative stress and mitochondrial apoptosis pathways, primarily involving the EndoG-IRE1/ATF6-CHOP signaling axis.